Project description:This sample was prepared with a modified version of the Tn4001 transposon commonly used in Mycoplasmas. This transposon is described to work more efficiently in Mycoplasma agalactiae and, by extension, in other Mycoplasma species. This sample was prepared transforming this bacteria with a newly designed transposon named pMTnGm-SynMyco to test its efficiency by Transposon Sequencing tracked by deep sequencing.
Project description:Here, we report a CRISPR/Cas12k-transposon-assisted genome engineering (CTAGE) method that allows for high-throughput site-specific mutagenesis in microbial genomes. Exploiting the powerful CTAGE technique, we construct a site-specific transposon mutant library focusing on all the possible transcription factors (TFs) in Pseudomonas aeruginosa, enabling comprehensive identification of essential genes and new factors for antibiotic resistance.
Project description:DNA transposon-based gene delivery vectors represent a promising new branch of randomly integrating vector development for gene therapy. For the side-by-side evaluation of the piggyBac and Sleeping Beauty systems - the only DNA transposons currently employed in clinical trials - during therapeutic intervention, we treated the mouse model of Tyrosinemia type I. with liver-targeted gene delivery using both transposon vectors. For genome-wide mapping of transposon insertion sites we developed a new Next Generation Sequencing procedure called Streptavidin-Based Enrichment Sequencing, which allowed us to identify approximately 1 million integration sites for both systems. We revealed that a high proportion of piggyBac integrations are clustered in hot regions and found that they are frequently recurring at the same genomic positions among treated animals, indicating that the genome-wide distribution of Sleeping Beauty-generated integrations is closer to random. We also revealed that the piggyBac transposase protein exhibits prolonged activity, which predicts the risk of oncogenesis by generating chromosomal double-strand breaks. Safety concerns associated with prolonged transpositional activity draw attention to the importance of squeezing the active state of the transposase enzymes into a narrower time window.
Project description:Precise genome engineering of human T cells, such as targeted integration of large transgenes into human T cells via homology-directed repair (HDR), has revolutionized adoptive cell therapies and deepened our understanding of T cell biology. Adeno-associated virus (AAV) vectors are used to deliver HDR templates into T cells, but the limited tropism of naturally occurring AAV serotypes remains a major barrier to efficient genetic manipulation in these cells. To overcome this challenge, we performed directed evolution of the AAV6 capsid and identified a new family of variants containing an Alanine-Proline-Arginine (APR) motif, with AAV.APR31 (also termed HONG31) emerging as one of the lead candidates. AAV.APR31 exhibited markedly enhanced transduction efficiency in human T cells, even at substantially lower vector doses compared to AAV6. Using genome-wide CRISPR-Cas9 knockout screens in human T and B lymphocytes, we identified CD7 as an essential gene required for AAV.APR31 transduction in T lymphocytes and NK cells. Structural analyses revealed that the APR substitution induced a spatial conformation change in the capsid surface, repositioning the VR-IV domain to improve accessibility and cooperative binding with CD7. We further demonstrate that AAV.APR31 enables robust transgene delivery, supports high-efficiency CRISPR-Cas9-mediated knock-in of large constructs at multiple genomic loci, and facilitates AAV-transposon-mediated DNA integration. Notably, AAV.APR31 has been successfully applied to generate a variety of therapeutic immune cell products, including conventional CAR-T cells, bispecific CAR-T cells, synthetic TCR-T cells, and CAR-NK cells. The development of these synthetic capsids expands the toolbox for precise genome engineering and holds strong translational potential for both basic research and next-generation human T and NK cell-based cellular immunotherapies.
Project description:Analysis of the episomal backbone's influence on gene expression. The first hypothesis tested in the present study is that the episomal EBNA vectors, which rely on the EBNA-1 oncoprotein for episomal maintenance, have a greater influence on the cells' expression profiles than S/MAR vectors. The second hypothesis tested was that when bacterial sequences are removed from the episomal vector backbone, the gene disturbance is minimal.
Project description:Adoptive cellular therapy using genetically engineered immune cells holds tremendous promise for the treatment of advanced cancers. While the number of available receptors targeting tumor specific antigens continues to grow, the current reliance on viral vectors for clinical production of engineered immune cells remains a substantial bottleneck limiting translation of promising new therapies. Here, we describe an optimized methodology for efficient CRISPR-Cas9 based, non-viral engineering of primary human T cells that overcomes key limitations of previous approaches. By synergizing temporal optimization of reagent delivery, reagent composition, and integration mechanism, we achieve targeted integration of large DNA cargo at efficiencies nearing those of viral vector platforms with minimal toxicity. CAR-T cells generated using our approach are highly functional and elicit potent anti-tumor cytotoxicity in vitro and in vivo. Importantly, our method is readily adaptable to current Good Manufacturing Practices (cGMP) and clinical scale-up, offering a near-term alternative to the use of viral vectors for production of genetically engineered T cells for cancer immunotherapy.